Net sulfatide synthesis, galactosylceramide sulfotransferase and arylsulfatase A activity in the developing cerebrum and cerebellum of normal mice and myelin-deficient jimpy mice

Net sulfatide synthesis, galactosylceramide sulfotransferase (EC 2.8.2.11) and arylsulfatase A (EC 3.1.6.1) activities were measured in two brain regions, cerebrum and cerebellum, of normal and jimpy mice during postnatal development. In normally myelinating mice, two phases of increasing rates of net sulfatide synthesis were observed, the first coinciding with oligodendrocyte proliferation and the second with myelination. Net sulfatide synthesis was quantitatively higher in the cerebellum than in the cerebrum. In both brain regions, the developmental patterns of net sulfatide synthesis were related to the activity patterns of both galactosylceramide sulfotransferase and arylsulfatase A. In jimpy mice, a neurological mutant showing hypomyelination in brain, the first phase of net sulfatide synthesis was preserved in both brain regions and galactosylceramide sulfotransferase and arylsulfatase A activities were normal up to 12 days. However, during the phase in which myelination occurred in controls, the net sulfatide synthesis in both brain regions of jimpy mice was zero or even negative. The sulfatide deficit was larger in the cerebellum than in the cerebrum. In both mutant brain parts, galactosylceramide sulfotransferase activity increased up to 12 days showing about 50% of the maximal activities observed in normal brain regions. Thereafter up to 15 days, enzyme activity decreased to about 25% of that of controls and remained low in both brain regions. The developmental patterns and the activities of arylsulfatase A were, however, normal in the cerebrum and cerebellum of jimpy mice. These results suggest that the enzyme activities and the developmental patterns of galactosylceramide sulfotransferase and arylsulfatase A as measured in vitro reflect to a high degree their functional activity in vivo. Furthermore, sulfatide degradation by arylsulfatase A seems to be important in regulating net sulfatide synthesis during normal and impaired myelination.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

Brain Slice Protein Degradation and Development

Protein degradation rates were measured in brain slices prepared from rats of various ages. This was done by adding the protein synthesis rate, determined by incorporation of a labeled precursor, and the net protein degradation rate, determined by measuring the changes with time of total free amino acids. These rates are about 30% higher than those previously calculated from data on protein synthesis rates and protein accumulation rates in vico. The protein degradation rates in brain slices diminish with age; i.e., 2-day cerebellum > 2-day cerebral hemisphere > 12-day cerebral hemisphere > young adult cerebral hemisphere. Protein degradation rates in slices from young brain are initially slightly higher than protein synthesis rates, resulting in a small net degradation with time. Unlike slices from adult brain, the protein degradation rates in slices from young brain decline only modestly with time for as much as 100 min of incubation. The characteristics of protein degradation in brain slices from young animals are roughly similar to some of the data calculated for protein degradation in vivi. suggesting that this system may prove useful for studying factors which control or affect brain protein degradation.     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

Brain DNA metabolism in myelin deficient mutant jp,jpmsd and qk mice

We studied metabolism of brain DNA in three myelin deficient mutants qk, jp and jp^msd mice. The DNA content, the in vivo incorporation of [¹⁴C]thymidine in DNA and the activity of acid DNase in tissues (cerebellum and cerebrum) from normal littermates and affected mice were compared. The results showed that neither the DNA content, the incorporation of [¹⁴C]thymidine in DNA nor the activity of acid DNase in brain were altered in qk affected mice. In jp^msd mice, however, the DNA content as well as the incorpation of thymidine in DNA were reduced in both cerebellum and cerebrum, but the activity of acid DNase was reduced in cerebrum only. In jp mice, although the DNA content was reduced in both cerebellum and cerebrum, the incorporation of thymidine in DNA and the activity of acid DNase were reduced in cerebrum only. The data suggest a) that in qk mutants DNA metabolism and hence cell (glial) proliferation is not affected; b) that in jp^msd mutants DNA synthesis, and thus the cell proliferation is reduced in cerebellum as well as in cerebrum of the affected mice and c) that in jp mutants the synthesis of DNA and the cell proliferation is reduced in cerebrum but not in cerebellum.     

A CONSIDERATION OF THE PERMEABILITY OF CARTILAGE TO INORGANIC SULFATE

On the basis of an examination of autoradiograms of knee-joints fixed so as to remove chondroitin sulfate or inorganic sulfate, or to minimize the loss of both, it is suggested that the cartilage is permeable to inorganic sulfate in vivo and in vitro. In vivo and in vitro, almost as rapidly as it enters the cartilage, inorganic sulfate is utilized by the cells in the synthesis of chondroitin sulfate. The net result is a continuing low concentration of inorganic sulfate in the cartilage.     

Heparan sulfate biosynthesis by embryonic tissues and primary fibroblast populations.

Fibroblasts from cornea, heart, and skin of day 14 embryonic chicks demonstrate the ability to make heparan sulfate-like polysaccharide when examined during the 10 hr period immediately following their removal from the embryo. Both the whole tissues from which these fibroblasts are isolated and the fibroblasts grown for 2–5 weeks in vitro also synthesize heparan sulfate. During their first few days in vitro, the three fibroblast populations display increasing rates of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporation into glycosaminoglycans and sharp fluctuations of those rates, yet the percentage of total [³⁵S]-sulfate incorporated into heparan sulfate-like polysaccharide and the distribution of this polysaccharide between cells and nutrient medium do not change significantly. During their first 48 hr in vitro, skin fibroblasts, but not those from cornea or heart, show steadily decreasing discrepancies between the proportions of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporated into heparan sulfate, suggesting a sharp decline in the synthesis of nonsulfated glycosaminoglycans. These data support the hypothesis of Kraemer than many cell-types in vivo may normally make heparan sulfate. The data largely eliminate the hypothesis that the biosynthesis of this polysaccharide is selectively stimulated as embryonic cells adapt to growth in vitro.     

Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

Ethanol and other CNS depressants decrease GABA synthesis in mouse cerebral cortex and cerebellum in vivo

GABA synthesis in mouse brain $\text{in vivo}$ was estimated by measuring the rate of GABA accumulation one hour after inhibition of GABA degradation using the selective and irreversible antagonism of GABA-transaminase by gabaculine. Using this method we found that acute and repeated ethanol administration lead to a potent depression of gabaculine induced enhancement of GABA levels in mouse brain cerebellum and cerebral cortex. Alcohol, in the absence of gabaculine had no effect on steady state GABA levels. These results demonstrate potent effects of ethanol on the dynamics of GABA metabolism which are compatible with a GABA like effect of ethanol.     

Increased synthesis of L-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, $\text{BALB}\text{cBy}$ mice have twice the level of enzyme activity in the cerebellum as do $\text{C57BL}\text{6J}$ mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of $\text{BALB}\text{cBy}$ cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.     

Effect of hyperphenylalaninemia induced during suckling on brain DNA metabolism in rat pups

We studied DNA metabolism (synthesis and degradation) in brain to investigate the effect of hyperphenylalaninemia induced in rats by treatment with PCPA or MPA plus PHE during suckling (4th–20th days of postnatal age) on cell proliferation and naturally occurring cell death. The incorporation of¹⁴C in DNA as percent of total radioactivity in the tissue, 30 min after administration of [¹⁴C]thymidine served as a measure of DNA synthesis in vivo, and the amount of radioactivity recovered in DNA as percent of total¹⁴C in the tissues of 21 day old rats, injected with [¹⁴C]thymidine on 2nd day after birth, indicated the turnover (degradation) of DNA. The results showed that the DNA content of cerebellum as well as cerebrum was reduced by treatment with PCPA plus PHE, while treatment with MPA plus PHE had no effect on DNA content in cerebellum but reduced the levels in cerebrum. Treatment with PCPA or MPA plus PHE reduced the synthesis of DNA in cerebrum of 11 day old rats but not in 21 day old rats, and the treatments did not affect DNA synthesis in cerebellum of either 11 or 21 day old rats. The turnover (degradation) of DNA was increased in both cerebellum and cerebrum from rats treated with PCPA plus PHE but MPA plus PHE treatment did not alter the DNA turnover either in cerebellu or in cerebrum. The activity of acid DNase was reduced in both cerebellum and cerebrum from 11 as well as 21 day old rats treated with PCPA plus PHE, but the enzyme activity was not altered in the tissues from rats of both ages treated with MPA plus PHE. The data thus indicate that in rats treated with PCPA plus PHE the reduction in cerebral DNA levels occurs due to reduced synthesis and/or increased turnover (degradation) of DNA but that the reduction in cerebellar DNA may occur only as a result of increased turnover (degradation), and that in rats treated with MPA plus PHE the reduction in cerebral DNA must occur due to reduced synthesis. This suggests that treatment of rats with PCPA plus PHE during suckling inhibits cell proliferation and/or increases naturally occurring cell death in both cerebellum and cerebrum while treatment with MPA plus PHE inhibits only cell proliferation and in cerebrum alone.     

THE EFFECTS OF PHENYLKETONURIC AND OTHER METABOLITES ON SULFATED GALACTOCEREBROSIDE SYNTHESIS IN VIVO AND IN CULTURE

Abstract— Sulfated galactocerebroside synthesis was examined in vitro in mouse spinal cord cultures. This system permitted the study of the effects of phenylketonuric metabolites upon synthesis of a specific myelin component, sulfatide, formed early in postnatal development in mice. A significant reduction of Na₂³⁵SO₄ incorporation into myelin sulfatide was observed when spinal cord cultures were grown in the presence of 1000 μm -l -phenylalanine and 500 μm -phenylpyruvate (51 and 700%, respectively). No reduction was observed with β-phenyllactate (300 μm and) phenylacetate (250 μm ). Light microscopy indicated that the phenylpyruvate and phenylalanine treated cultures were less extensively myelinated compared to control and β-phenyllactate or phenylacetate treated cultures. The reduction of sulfatide synthesis by phenylpyruvate was shown to be reversible. Intracerebral bilateral injections (8 μg) of l -phenylalanine, phenylpyruvate, α-ketobutyrate, α-ketoisocaproate, α-ketoisovalerate, β-phenyllactate, and phenylacetate in mice 8–15 days old, followed by i.p. administration of radioactive sulfate, resulted in significantly reduced incorporation (all P < 0.05) of sulfate into brain sulfatides with all compounds tested with the exception of β-phenyllactate and phenylacetate. In adult mouse, phenylpyruvate treatment also resulted in a significant decrease in labelling of brain sulfatide. The effects of phenylpyruvate and other metabolites upon pyruvate oxidation in mouse brain homogenates were examined by measuring ¹⁴CO₂ release from [1-¹⁴C]pyruvate. Both phenylpyruvate and α-ketoisocaproate at 1 × 10^-3 resulted in a decrease in ¹⁴CO₂ produced, while phenylacetate and β-phenyllactate had no effect. Sulfate incorporation into sulfatide was reduced by α-ketoisocaproate and phenylpyruvate, and to a lesser extent by phenylalanine, α-ketobutyrate, and α-ketoisovalerate. Phenyllactate and phenylacetate had no effect, either in vivo, or in culture. This order of effectiveness may be related in part to the effects of these compounds on pyruvate oxidation.     

Regional distribution of homocarnosine and other ninhydrin-positive substances in brains of malnourished monkeys

—Eight male monkeys (Macaca nemestrina) aged 6–9 months were divided into two groups and fed either an adequate protein diet (20% casein) or a protein deficient diet (2% casein). After 3- 5 months of receiving the low protein diet, the malnourished monkeys showed extensive fatty metamorphosis of the liver cells, distorted patterns of plasma and hepatic free amino acid pools, and other features consistent with the diagnosis of protein-calorie malnutrition. Examination of the cerebrum, cerebellum and brain stem in the malnourished animals revealed profound accumulation of 3-methylhistidine, histidine and homocarnosine in all three regions. For histidine, the cerebral, cerebellar and brain stem levels in the protein deficient animals increased by 145, 104 and 101 per cent over levels observed in corresponding regions of the brain in well-fed monkeys. Similarly, there were significant elevations in homocarnosine contents of the cerebrum (+ 99 per cent), cerebellum (+ 140 per cent) and brain stem (+ 146 per cent) in comparison to levels in control animals. In contrast, the levels of valine, serine and aspartic acid were markedly reduced in all three brain areas in the malnourished animals. Protein-calorie deficiency also produced reductions in the brain levels of taurine, glutamic acid, isoleucine, leucine and threonine which varied in magnitude in the three major regions of the brain examined. These biochemical alterations which may in part underlie some of the psychomotor changes often observed in protein-calorie malnutrition, were discussed not only in relation to the role of amino acids as precursors for the synthesis of neuroregulatory substances but also with due regard to the possibility that some of these ninhydrin-positive substances such as GABA, homocarnosine, glycine and the dicarboxylic amino acids may possess neuroexcitatory or inhibitory properties in various parts of the central nervous system.     

Protective Effects of Selenium on Cadmium-Induced Brain Damage in Chickens

Selenium (Se) is an important dietary micronutrient with antioxidative roles. Cadmium (Cd), a ubiquitous environmental pollutant, is known to cause brain lesion in rats and humans. However, little is reported about the deleterious effects of subchronic Cd exposure on the brain of poultry and the protective roles on the brain by Se against Cd. The aim of this study was to investigate the protective effects of Se on Cd-induced brain damage in chickens. One hundred twenty 100-day-old chickens were randomly assigned to four groups and were fed a basal diet, or Se (as 10 mg Na₂SeO₃/kg dry weight of feed), Cd (as 150 mg CdCl₂/kg dry weight of feed), or Cd?+?Se in their basic diets for 60 days. Then, concentrations of Cd and Se, production of nitric oxide (NO), messenger RNA (mRNA) level and activity of inducible NO synthase (iNOS), level of oxidative stress, and histological and ultrastructural changes of the cerebrum and cerebellum were examined. The results showed that Cd exposure significantly increased Cd accumulation, NO production, iNOS activities, iNOS mRNA level, and MDA content in the cerebrum and cerebellum. Cd treatment obviously decreased Se content and antioxidase activities and caused histopathological changes in the cerebrum and cerebellum. Se supplementation during dietary Cd obviously reduced Cd accumulation, NO production, mRNA level and activity of iNOS, oxidative stress, and histopathological damage in the cerebrum and cerebellum of chickens. It indicated that Se ameliorates Cd-induced brain damage in chickens by regulating iNOS-NO system changes, and oxidative stress induced by Cd and Se can serve as a potential therapeutic for Cd-induced brain lesion of chickens.     

Influence of Proline on Rat Brain Activities of Alanine Aminotransferase,Aspartate Aminotransferase and Acid Phosphatase

Hyperprolinemia type II (HPII) is an autosomal recessive disorder caused by the severe deficiency of enzyme ¹-pyrroline-5-carboxylic acid dehydrogenase leading to tissue accumulation of proline. Chronic administration of Pro led to significant reduction of cytosolic ALT activity of olfactory lobes (50.57%), cerebrum (40%) and medulla oblongata (13.71%) only. Whereas mitochondrial ALT activity was reduced significantly in, all brain regions such as olfactory lobes (73.23%), cerebrum (70.26%), cerebellum (65.39%) and medulla oblongata (65.18%). The effect of chronic Pro administration on cytosolic AST activity was also determined. The cytosolic AST activity from olfactory lobes, cerebrum and medulla oblongata reduced by 75.71, 67.53 and 76.13%, respectively while cytosolic AST activity from cerebellum increased by 28.05%. The mitochondrial AST activity lowered in olfactory lobes (by 72.45%), cerebrum (by 78%), cerebellum (by 49.56%) and medulla oblongata (by 69.30%). In vitro studies also showed increase in brain tissue proline and decrease in glutamate levels. In vitro studies indicated that proline has direct inhibitory effect on these enzymes and glutamate levels in brain tissue showed positive correlation with AST and ALT activities. Acid phosphatase (ACP) activity reduced significantly in olfactory lobes (40.33%) and cerebrum (20.82%) whereas it elevated in cerebellum (97.32%) and medulla oblongata (76.33%). The histological studies showed degenerative changes in brain. Following proline treatment, the animals became sluggish and showed low responses to tail pricks and lifting by tails and showed impaired balancing. These observations indicate influence of proline on AST, ALT and ACP activities of different brain regions leading to lesser synthesis of glutamate thereby causing neurological dysfunctions.     

Effect of subchronic exposure to arsenic on levels of essential trace elements in mice brain and its gender difference

The interactions of toxic metals with essential metals may result in disturbances in the homeostasis of essential elements. However, there are few reports about toxic effect of arsenic (As) on the levels of essential trace elements in the central nervous system. To investigate whether subchronic exposure to As disturbs levels of main essential trace elements in the brain of mice and whether the gender difference in the response to As are altered, the concentrations of As, Iron (Fe), copper (Cu), selenium (Se), zinc (Zn) and Chromium (Cr) in the cerebrum and cerebellum of mice exposed to As subchronically were examined by inductively coupled plasma-mass spectrometry (ICP-MS). The gender difference in the changed levels of these essential trace elements was also statistically analyzed. The concentration of As was significantly higher in the cerebrum or cerebellum of mice exposed to As than that in control group (P < 0.05). It indicates that As can accumulate in brain of mice after subchronic exposure. The concentrations of Fe, Se and Cr in the cerebrum or cerebellum were significantly lower in mice exposed to As than those in control group (P < 0.05). On the contrary, the concentration of Cu in the cerebrum or cerebellum was significantly higher in mice exposed to As (P < 0.05). Our results indicate that subchronic exposure to As may decrease the levels of Fe, Se and Cr or increase the level of Cu in the brain of mice. Moreover, the significant gender difference was found relative to the effect of As on concentration of Se in cerebrum and concentrations of Cu and Se in cerebellum of mice. Therefore, more experiments are required to further understand mechanisms whereby As interacts with essential elements in brain and induces the gender difference.     

Diffusion of intracerebrally injected [1-14C]arachidonic acid and [2-3H]Glycerol in the mouse brain

[2-³H]Glycerol and [1-¹⁴C]arachidonic acid were injected into the region of the frontal horn of the left ventricle of mice and were distributed rapidly throughout the brain. After 10 sec, most of the radioactive fatty acid was found in the hemisphere near the injection site; after 10 min, it was recovered in similar proportions in the cerebellum and brain stem. [2-³H]Glycerol showed a heterogeneous distribution, with most of the label remaining in the left hemisphere even after 10 min. On a fresh weight basis, cerebrum, cerebellum, and brain stem were found to contain similar amounts of labeled glycerol. However, the amount of [1-¹⁴C]arachidonate in cerebrum was only 50% of that recovered from cerebellum or brain stem. Brain ischemia or a single electroconvulsive shock reduced the spread of the label, producing an accumulation of radioactivity in the injected hemisphere, except for an increase in [2-³H]glycerol in the brain stem during ischemia. Despite the significant decrease in available precursor in the cerebellum and brain stem after electroshock, the amount of label incorporated into lipids was not altered in these areas and only slightly diminished in the cerebrum.     

Connexin43 expression during Xenopus development

The polypyrimidine tract binding protein (PTB) and its recently discovered homologue brain-enriched PTB (brPTB) are RNA binding proteins involved in the control of alternative splicing. We have characterized expression patterns of the PTB and brPTB in course of mouse brain development, using mRNA in situ hybridization. PTB is expressed in choroid plexi and ependyma at all the stages of development and temporarily in the mantle layer of migrating neuroblasts of fore-, mid- and hindbrain and in the external granular layer of cerebellum. In the neurons of adult mouse cerebrum and cerebellum expression of PTB is undetectable. In contrast to this, brPTB is expressed ubiquitously in neuroblasts of various parts of embryonic brain and in the differentiated neurons of postnatal cerebrum and cerebellum. brPTB mRNA is not observed in choroid plexi and ependymal layer. Thus, in the embryonic brain expression patterns of PTB and brPTB overlap, but in the course of brain development the patterns become complementary to each other.     

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.